There are a number of assay systems available for detection and quantitation of analytes, particularly analytes of biological interest. Current assay systems include enzyme immunoassay (EIA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). Among the analytes frequently assayed with such systems are: 1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed in urine as a marker of human pregnancy; 2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus and hepatitis virus; 3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as antibody to the bacterium Heliobacter pylori and to human immunodeficiency virus (HIV); 4) enzymes, such as aspartite aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; 5) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer; 6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroine, and marijuana; and 7) environmental pollutants such as pesticides and aromatic hydrocarbons and vitamins.
Such systems are frequently used by physicians and medical technicians for rapid in-office diagnosis and therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by patients themselves for at-home or on-site monitoring of such conditions and disorders.
Among the most popular of such assay systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody. The use of immunoassays as a means of testing for the presence and/or amount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (Singer et al., 1956).
Development of the first radioimmunoassay by Rosalyn Yalow and Sol Berson (1959) set the stage for measurement of a wide variety of hormones in biological fluids by binding the hormone specifically and with high affinity to antibodies developed in animals against the hormone in question. The assay developed by Drs. Yalow and Berson employed antibodies formed against the protein hormone, insulin, and utilized a radiolabeled form of insulin as the marker, or “reporter” hormone. Antibodies became a useful way to “capture” a specific hormone from biological fluids and under conditions of constant antibody concentration and with some easily detected source of labeled hormone (usually radioactively labeled; hence the name “radioimmunoassay”) the amount of hormone “captured” from the biological fluid could be quantified by comparison to known concentrations of the hormone in similar conditions. In practice of the art, known amounts of (unlabeled) hormone, (insulin in the example) were allowed to compete for binding to the antibody with a known and fixed amount of I131 labeled insulin. The radiolabeled form of hormone, and the amount of antibody were held constant while the amount of unlabeled hormone was varied. This was the basis of a “standard curve” from which the amount of radioactive label that bound to the antibody varied inversely with the amount of unlabeled hormone. Comparison of the mass of unlabeled hormone required to displace a given amount of labeled hormone could then be used to estimate mass of an sample hormone. Separation of the fractions which were unreacted with the antibody (unbound) was carried out by a variety of chemical separation methods. In the original teaching of Yalow and Berson (1959), separation of the antibody-bound fractions of insulin from the unbound (free) fractions of insulin was carried by electrophoresis. Subsequent to their report, many means of separating bound from free fractions have been utilized, including column chromatography, salt or organic solvent precipitation of the protein (antibody), double antibody (in which the gamma globulin fraction of the species immunized against the hormone is then introduced to a different species to create an anti-antibody, or second antibody, and solid phase, in which the antibody is held by electrostatic forces to a solid interphase such as the inner wall of a test tube, flat disc, or elongate stick (dipstick) and separation of bound from free requires simple physical separation of the solid phase from the liquid phase containing the free fractions. A variant of the technique of radioimmunoassay involved coupling small, non-immunogenic molecules to larger, highly immunogenic molecules, such as bovine serum albumen (BSA), thyroglobulin (TG), or keyhole limpet hemocyanin (KLH) and stimulation of antibodies that recognized the smaller, non-immunogenic, portion of the hapten molecule. This modification of the technique permitted quantification of small hormones, such as steroids and prostaglandins.
While radioimmunoassay is a very useful tool for conducting research and for certain clinical applications, it has several drawbacks as far as practical management of endocrine or other hormonal states. A major drawback is the use of an antibody as a “capture protein.” Development of polyclonal antibodies is accomplished by administering the hormone to an animal that regards it as foreign and develops antibodies against it. The process is very much trial and error and involves the use of a number of animals and screening of the antibody before determination of its usefulness. Once an ideal polyclonal antibody preparation has been obtained, the animal's plasma must be harvested and husbanded carefully, for once the animal dies, the supply of that particular antibody is lost forever.
The act of mounting an immune attack against a foreign protein and producing antibodies is actually a mixture, or collection of antibodies (hence the term “polyclonal antibody”), each of which is directed against a particular amino acid sequence called an epitope. A refinement of this process involves the production of monoclonal antibodies. Monoclonal antibodies are derived by collecting individual spleen cells from animals immunized by administration of a foreign protein and culturing the lymphocytes in vitro. The cells are then screened to determine their binding characteristics, and those cells that possess appropriate binding are then cloned and maintained as an antibody-specific, continuous cell line. Thus, once appropriate cell cultures are obtained, they may be kept essentially indefinitely, thereby obviating one of the negative aspects of polyclonal antibodies.
However, monoclonal antibodies also have some drawbacks. For one thing, they are so specific as to be a detriment in some cases. Monoclonal antibodies are directed against amino acid sequences (epitopes) that are often common features of the tertiary structure of proteins. In this case the monoclonal antibodies are really not specific as one might believe at first. This drawback can be overcome by very stringent screening and validation of the assays utilizing monoclonal antibodies, but greater effort is often required. Additionally, monoclonal antibodies tend to be monovalent, which may restrict hierarchical or sandwich type coupling to other molecules for the purpose of separation or of amplification of the reporter signal.
Immunoassays fall into two principal categories: “sandwich” and “competitive,” according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. Generally, the sandwich immunoassay calls for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a detectable label or a disclosing reagent, such as dyed latex or a radioisotope. This mixture is then applied to a chromatographic medium containing a band or zone of immobilized antibodies to the analyte of interest. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs and the bound labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results. Examples of sandwich immunoassays performed on test strips are described by U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by reference.
In competitive immunoassays, the label is typically a labeled analyte or analyte analogue which competes for binding of an antibody with any unlabeled analyte present in the sample. Competitive immunoassays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are those disclosed by U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all of which are incorporated herein by reference.
Although useful, currently available immunoassay techniques have a number of disadvantages. For example, because antibodies and other immunoassay reagents are susceptible to environmental conditions, at-home or on-site methods are problematic. Further, antibodies continue to be expensive to produce. Accordingly, it would be advantageous to employ an analyte assay system with all of the advantages of an immunoassay, but which is free of the inherent disadvantages traditionally associated with such immunologically based systems.
In addition to immunoassays, protein-binding assays have been utilized to quantitatively detect various analytes within a sample. These assay systems utilize a protein, such as a protein receptor, which is specific for a particular analyte target. Unfortunately, because these systems utilize the entire protein and the protein may have binding sites for more than one target analyte, there can be problems with cross-reactivity and assay accuracy. For example, in angiotensin II SPA receptor binding assays, the whole angiotensin membrane protein receptor is immobilized on a bead. This bead-receptor complex is then contacted with a sample, binding any angiotensin present in the sample.
Determination of optimum breeding time is important to the success of breeding domestic species. For example, the ability to rapidly and accurately measure equine ovarian steroid hormone estradiol (E2), in the field, would greatly benefit veterinarians in assisting managers of equine breeding farms in making breeding management decisions. The literature is replete with reference to early pregnancy failure in a variety of domestic species, e.g. sheep, 20 to 30% (Edey, 1969; Hanley, 1961; Nancarrow, 1994), goats, 6 to 42% (Kidder et al., 1954; Diskin et al., 1980), pigs, 20 to 30% (Bolet, 1986), cattle, 8 to 42% (Pope et al., 1985) and mares, 15 to 25% (Ginther, 1986; Villahoz et al., 1985; Ball et al., 1986; Ball et al., 1987). A major component of these losses represents errors of fertilization and/or exchange of genetic information (Hunter, 1994; King, 1990) or errors in the interaction between the maternal uterine unit and the developing conceptus (Bazer et al., 1986) even if successful breeding has occurred. With such losses inherent to the reproductive process, it is critical for veterinarians and managers to select the optimal time for breeding to maximize the potential for a successful pregnancy.
Current on-site methods for selecting the optimal time of breeding in the equine industry include the observation of behavioral interactions between the mare and stallion (teasing), rectal palpation and/or ultrasound to monitor ovarian follicular growth, as well as obligatory breed registry regulations. Teasing refers to the observation of the mare's behavior to the presence of a stallion. Distinct behavioral patterns are observed during each phase of the equine estrous cycle (Ginther, 1992). Palpation involves the insertion of the arm into the mare's rectum to manually determine ovarian follicular activity. Often, ultrasound viewing of ovarian follicular activity is also utilized. Accurate record of teasing and palpation/ultrasound data are essential for good breeding management.
Predicting optimal breeding time is also important because the nature of the breeding business adds constraints to successful conception. Most breeders do not keep stallions on their farms, and access to popular stallions requires scheduling and transportation of the mare to the stallion at a predetermined time (reserved booking date/time). This process adds substantial cost and financial risk and, therefore, increases the value of a tool that can predict ovulation. Furthermore, the arbitrary January 1 birth date employed by many breed registries requires that breeding and pregnancy occur as early in the breeding season as possible. Given a gestation length of 340 days, on average (Jeffcoat, 1972), early pregnancy results in birth as early in the following January as possible, creating a more mature, market valuable, horse for sale or training. According to the TBH Market Watch (1997), January foals were sold for 25% more compared to foals born in other months (i.e., February through July).
While predicting optimal breeding time is advantageous, few managers have the skill to accurately perform rectal palpation and even fewer have access to ultrasound. Veterinarians customarily charge managers for each farm visit and for each mare which they have to palpate and/or ultrasound. Moreover, traditional on-site breeding management practices (teasing, rectal palpation and/or ultrasound) cannot determine if a pre-ovulatory sized follicle will ovulate or regress. Accordingly, any technology which would allow the pre-selection of mares which require veterinary attention would be more efficient and economical.
The ovarian steroid hormone estradiol (E2) is a reliable indicator of the time of ovulation in mammals. During the breeding season, E2 is a critical hormone for normal follicular maturation, uterine endometrial development and ovulation in mares, as well as other mammals (Lipner, 1988). Furthermore, E2 exhibits a distinct secretory pattern during the estrous cycle during the breeding season. Mares are seasonal breeders with an annual reproductive cycle consisting of four phases: the breeding season (late spring to summer), the autumnal transition to Anestrus (late summer and fall), Anestrus (winter) and the vernal transition from Anestrus to the breeding season (late winter and spring) (Sharp, 1980). The breeding season is characterized by repeated estrous cycles, providing multiple, successive opportunities to become pregnant. Concentrations of E2 in blood are low (5 to 10 pg/ml) during the diestrus period; increase dramatically (30 to 70 pg/ml) in the 3 to 4 days preceding ovulation; peak 1 day prior to ovulation, on average; then decline to low diestrus concentrations (Pattison et al., 1974). Estrous cyclicity continues until either pregnancy results or declining day length initiates the transition into anestrus. Autumnal transition is not very well characterized. Briefly, a decline in hypothalamic-pituitary support, including GnRH (Strauss et al., 1979), LH and FSH secretion (Ginther, 1992), results in a progressive disruption in ovarian follicular growth and steroid hormone production. Anestrus begins when GnRH secretion reaches low, unvarying levels, LH and FSH secretion cease and ovarian follicular activity ceases (Ginther, 1992). Increasing day length in late winter initiates an increase in GnRH secretion and FSH secretion resulting in increased follicular growth (Ginther, 1992). An average of between 3 and 4 large, pre-ovulatory size follicles develop but fail to ovulate during this period (Tucker et al., 1993). These large, anovulatory follicles create considerable confusion as they are similar to ovulatory follicles in diameter, but they are unaccompanied by an increase in LH and they do not ovulate. Furthermore, these anovulatory follicles do not produce E2. LH secretion and E2 secretion do not recommence until immediately prior to the first ovulation of the year (the start of the breeding season) (Sharp et al., 1997).
Currently, veterinarians and breeding managers can determine E2 levels in mares only by submitting blood samples to a diagnostic laboratory. This process is costly and results are usually not available for 24 hours to one week, depending on the lab. Present commercial assay systems for E2 require several hours of incubation and expensive detection systems. Such assays utilize radioactive substances or hazardous chemicals and complicated procedures, neither of which is compatible with on-site use. For example, U.S. Pat. No. 5,460,976 teaches an luminescence assay for measuring oestradiol in the blood sample of an equine using two antibodies, an antibody against human oestradiol and an antibody against human FSH. Unfortunately, in view of the extreme sensitivity of the immunologic components to both environmental condition and chemical environment, this method also does not lend itself to on-site use. Accordingly, the ability to rapidly and accurately measure E2, in the field, would greatly benefit veterinarians in assisting managers of equine breeding farms in making breeding management decisions.